Title

Author

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Sangeeta Dhaubhadel

Abstract

Isoflavonoids are specialized metabolites, almost exclusive to the legume family of plants. They are actors in symbiosis with nitrogen-fixing bacteria and in plant stress response. Isoflavonoids are noted for their human health benefits. Isoflavonoid content in legumes has proven to be a complex trait. The goal of the present research is to determine the mechanisms underlying isoflavonoid biosynthesis in soybean.

The first approach was to unravel the genetic factors of isoflavonoid biosynthesis. A branch-point enzyme of the phenylpropanoid pathway, chalcone isomerase (CHI), catalyzes the reaction producing flavanones, the nucleus for many downstream metabolites such as isoflavonoids. I identified twelve soybean CHI genes, including five new members. The evolutionary history of the family shows that the enzymatic fold evolved from being catalytically inactive to being a chalcone-to-flavanone isomerase. Four CHIs in soybean were identified with the latter functionality. The CHIs showed differential temporal and spatial expression, pointing to the potential function of CHI1A in soybean seed isoflavonoid production. On the molecular level of organization, the long-postulated model of a subcellular isoflavonoid enzyme complex forming at the surface of the endoplasmic reticulum (ER) was substantiated in the present study. Here, I identified key players in the ‘metabolon’, established in planta subcellular localization, and investigated protein-protein interaction. The results suggest that isoflavone synthase (IFS), a cytochrome P450, is a nucleating metabolic center at the surface of the ER, interacting with upstream pathway enzymes. Finally, a transcriptomic study was undertaken to find genetic elements linked with isoflavonoid content variation in four soybean cultivars. The results suggest that competing branches of the phenylpropanoid pathway are combinatorially regulated to coordinate flux into isoflavonoid biosynthesis. The candidate genes encode enzymes in the overlapping pathways, several transcription factors, metabolic transporters and more.

Study of the CHI gene family and isoflavonoid biosynthesis has provided us with new insights into production and regulation of this important plant natural product. This knowledge can facilitate the manipulation of metabolic content and composition in legumes, and introduction of the isoflavonoid pathway into non-legume crops.